Lab Entry 8 – DNA Plant Extraction via Modified Alexander et al. Tube Protocol
This lab is the continuation of the past field trips that were taken in which various plant samples were collected around the Bay Area. The samples assigned to me originated from Abbotts Lagoon in Point Reyes. For all of the labeling, the code ‘PRA’ was used followed by the 01-05 for each of the five samples used during the lab.
1.5 mL centrifuge tubes were labeled PRA01, PRA02, etc., for the five plant samples collected from my assigned location. A small amount of leaf tissue was added to each of the tubes accordingly along with three 3.2-mm stainless steel beads and were placed in a tube rack along with some of the other students’ designated plant samples as well. The rack was then loaded into a modified reciprocating saw rack mount and was reciprocated on speed 3 for approximately 40 seconds.
The tubes were then spun down in the centrifuge for 15-20 seconds to pull the plant particles to the bottom of the tube. Approximately 434 μl of preheated grind buffer was added to each of the tubes and incubated at 65 °C for 10 minutes in a water bath, mixing the tubes every three minutes or so.
Once finished in the bath, 130 μl 3M pH 4.7 potassium acetate was added to each of the tubes and inverted to mix before being placed on ice to incubate for 5 minutes.
After incubating, tubes were centrifuged at maximum force for 20 minutes. New 1.5 mL tubes were labeled with the same sample ID as before. Any supernatant at the top of the centrifuged tubes was transferred out using pipettes, carefully avoiding transferring any of the precipitate. To these new tubes, 1.5 volumes of binding buffer was added for each of the tubes based on the volume of supernatant that was acquired. I added anywhere from 400 to 500 μl of buffer to the tubes with one tube needing less due to a lower volume of supernatant being acquired.
650 μl of the solution (or all of the solution if the volume was less than 650 μl) from the tubes was transferred to Epoch spin column tubes and were centrifuged for 10 minutes at 15,000 rpm in order to collect the DNA in the silica membrane filter as the buffer drained through the filter into the collection portion of the tube below. The buffer left in the tube was discarded and the filtered portion was added back to the tube. The tubes were then centrifuged again for another 10 minutes, and again, any liquid at the bottom of the tube was discarded.
DNA bound to the silica membrane filter was washed by adding 500 μl of 70% EtOH to the column and centrifuged for 8 minutes which would pass to the collection tube. The flow-through was discarded. Tubes were centrifuged again for approximately 5 minutes to remove any residual ethanol. The collection tubes were discarded and the columns were placed in sterile 1.5-mL microcentrifuge tubes that were also labeled with the plant codes on them.
To the new tubes, 100 μl preheated (65 °C) pure sterile water was added and were let to stand for about 5 minutes. After standing, the tubers were placed in the centrifuge and ran for 2 minutes at 15,000 rpm to elute the DNA. The tubes for the class were then all collected to be stored for the next lab session.
